During FY2014: 1) With Italian collaborators, we developed a test for CJD using nasal brushings because definite diagnosis of sporadic CJD in living patients remains a challenge. We previously described a test that detects the specific marker for CJD, the prion protein PrP(CJD), by means of RT-QuIC testing of cerebrospinal fluid has a sensitivity of 80 to 90% for the diagnosis of sporadic CJD. To improve on the sensivity and practicality of CJD diagnosis in living patients, we assessed the accuracy of RT-QuIC analysis of nasal brushings from olfactory epithelium. Our neurologist collaborators collected olfactory epithelium brushings and cerebrospinal fluid samples from patients with and patients without sporadic CJD and tested them using RT-QuIC, an ultrasensitive, multiwell plate-based fluorescence assay involving PrP(CJD)-seeded polymerization of recombinant PrP into amyloid fibrils. The RT-QuIC assays seeded with nasal brushings were positive in 30 of 31 patients with CJD (15 of 15 with definite sporadic CJD, 13 of 14 with probable sporadic CJD, and 2 of 2 with inherited CJD) but were negative in 43 of 43 patients without CJD, indicating a sensitivity of 97% and specificity of 100% for the detection of CJD. By comparison, testing of cerebrospinal fluid samples from the same group of patients had a sensitivity of 77% and a specificity of 100%. Nasal brushings elicited stronger and faster RT-QuIC responses than cerebrospinal fluid. Individual brushings contained approximately 10(5) to 10(7) prion seeds, at concentrations several orders of magnitude greater than in cerebrospinal fluid. We concluded that RT-QuIC testing of olfactory epithelium samples obtained from nasal brushings was accurate in diagnosing CJD and indicated substantial prion seeding activity lining the nasal vault. 2) We assisted collaborators at Colorado State University in modifying of RT-QuIC assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans. 3) We also collaborated with researchers at CSU to adapt the RT-QuIC assay to the detection of prion seeding activity in the saliva of deer infected with CWD. CWD is an efficiently transmitted prion disease of cervids. Rapid in vitro detection of CWD (and other) prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA) and the presence of inhibitors in these complex biological materials that hamper detection. We used real-time quaking induced conversion (RT-QuIC) to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3%) diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%). In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1%) of the above saliva samples. Bioassay in TgCerPrP mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis we described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination.